A lypohilised vial of GL67A before resuspension. The lipid forms a dense white powder that is stored under Nitrogen.

GL67A/pGM169 is the combination of cationic liposome (GL67A) and plasmid DNA expressing CFTR (pGM169) that the consortium is currently assessing in our ongoing clinical trial programme. Aerosolisation of a plasmid DNA vector to the lungs of patients represents an enormous challenge and plasmid DNA cannot be efficienctly delivered to the airways on its own.

Therefore we combine the negatively charged plasmid with positively charged fatty liposomes (GL67A) and these form very small, tighly bound particles which are stable enough to withstand aerosolisation and can be taken up be the cell membane of cells in the respiratory tract.

Following uptake by the cells plasmid DNA is transported to the nucleus where it starts to express its copy of the CFTR gene.

A western blot to detect CFTR protein expression from human cell lines. pGM169 is in the two right hand lanes.

A map of our clinical trial plasmid pGM169

The plasmid vector pGM169 was created in Oxford in mid 2006 during our pre-clinical development work. In a number of studies this plasmid easily established itself as our most promising candidate for clinical testing.

The CFTR cDNA was codon optimised to maxmise the efficiency of CFTR protein production (the natural human CFTR sequence is actually really inefficient). This cDNA sequence was coupled with our groundbreaking hCEFI promoter which provides the longest duration of CFTR activity that we have ever observed in our pre-clinical models (Hyde et al. 2008).

In addition, by using technology developed initially by the biotech company Invivogen, we were able to make the plasmid completely devoid of any CG dinucleotides which have been associated with causing an inflammatory response in-vivo.

A single nasal administration of the GL67A/pDNA formulation was safe, directed vector-derived mRNA expression, and produced an overall ~20% correction of the CFTR Cl- channel defect in the nose (Zabner et al. 1997). Of all the cationic lipids available for non-viral gene transfer, Genzyme Lipid GL67A is still the only non-viral GTA that has been successfully aerosolized to the lungs of patients in phase I clinical trials (Alton et al. 1999) & (Ruiz et al. 2001).

After delivery to the lungs of CF patients via jet nebuliser vector-derived transgene mRNA was detected in four out of nine bronchoscopic lung samples (Ruiz et al. 2001). Using the same formulation Alton reported partial correction of the CF Cl- transport defect in the nose, trachea and lower airways of treated CF subjects (Alton et al. 1999), providing proof-of-principle for correction of CFTR-mediated Cl- secretion following gene transfer to the lung.

The Consortium trials that are underway represenent the third time that GL67A has been used in a gene therapy clinical trial for CF, but the decision to proceed was reached only after an extensive programme of testing, where GL67A out-performed many new and established GTAs in mouse and sheep models. Based on these studies we beleive GL67A still represents the 'Gold Standard' for aerosol delivery of a non viral vector to the airways.

Making a clinical product

Excess large scale (2 litres+) high concentration pGM169

Nebuliser attached to a ventilator test rig

Much has changed since the last clinical trials were undertaken for GL67/pDNA complexes. Therefore the Consortium had to put a lot of effort into making the lipid DNA combination suitable for a 21 Century clinical trial.

The scale of the planned clinical trials is such that production of GL67A and pGM169 had to be contracted out to biotech firms. The plasmid is being manufactured by VGXi in Houton, Texas. In order to conduct our planned multidose clinical trial, so much pGM169 is required that it will become the most abundant plasmid molecule ever to have been produced for human clinical use.

Aerosol technology has also changed much in recent years so even the process of selecting an appropriate nebuliser for the clinical trial become a vital research project requiring several months of testing (Davies et al, 2008). Finally a new method of mixing the lipd and DNA was developed to allow for complete control a reproducibility of every dose (Davies et al. 2010).